Download using makeoligos_2b

using makeoligos_2b.m
command line syntax:
[oligoout,seq,tm_out,overlapbeg_out,overlapend_out]=makeoligos2b(protein,prefix,suffix,readingframe,re1,re2,org)
;
protein:
prefix:
suffix:
readingframe:
re1 and re2:
org:
the protein sequence, no gaps
constant DNA sequence to be appended to the 5' end of the gene
constant DNA sequence to be appended to the 3' end of the gene (both
prefix and suffix will be the coding sequence for the gene)
1, 2 or 3, the reading frame of the protein once the prefix is added.
If your prefix is divisible by 3, then readingframe=1
restriction enzymes used for cloning. The program will inform you if
the design generates internal restriction sites.
'e' or 'y', to indicate whether E. coli or yeast codon preferences will
be used.
notes:
 the program designs sequences with the following properties:
o the 3' base must be a G or C
o at least 3/5 of the 3' bases must be G or C
o overlap Tm is 54 or 56 °C, using the formula Tm = (#GC * 4) + (#AT * 2)
o maximum oligo length is 45
o optimal codons are used when possible, otherwise, a second-best codon is
used.
 the 3'most oligo must be antisense in order for the PCR to work (theoretically).
However, the program doesn't take this into account. Therefore, I recommend
(manually) building an anti-sense oligo against the constant suffix sequence that
has the properties described above.
 the output indicates number of occurrences of re1 and re2, including the instances
specified in prefix and suffix. If these values are greater than 1, then there is
an extra site somewhere in the gene. You have to change this by manually altering
the offending oligos, sticking to the criteria described above. Codon tables with
preferences for yeast and E. coli are attached.
an example:
here is the first sequence in the WW alignment:
>> aln(1,:)
ans =
PLP-------PG---WEIKIDPQTGWPFFVDHNSRTTTWNDPRVP
to get it without gaps, use:
>> aln(1,find(aln(1,:)~='-'))
ans =
PLPPGWEIKIDPQTGWPFFVDHNSRTTTWNDPRVP
[oligoout,seq,tm_out,overlapbeg_out,overlapend_out]=makeoligos2b(aln(1,find(aln(1,:
)~='-')),'actgacggatcc','ggtgaattcgtgggcg',1,'ggatcc','gaattc','e');
or
[oligoout,seq,tm_out,overlapbeg_out,overlapend_out]=makeoligos2b('PLPPGWEIKIDPQTGWP
FFVDHNSRTTTWNDPRVP','actgacggatcc','ggtgaattcgtgggcg',1,'ggatcc','gaattc','e');
the output looks like:
actgacggatccccattgccaccaggttgggaaattaaaatcgac
ggccaaccagtttgtgggtcgattttaatttcccaac
ccacaaactggttggccattttttgttgatcacaactccaggacc
ggacccttgggtcgttccaagtagtggtcctggagttgtgatc
gaacgacccaagggtcccaggtgaattcgtgggcg
actgacggatccccattgccaccaggttgggaaattaaaatcgacccacaaactggttggccattttttgttgatcacaactccaggacc
gaacgacccaagggtcccaggtgaattcgtgggcg
gttgggaaattaaaatcgacccacaaactggttggcc
gatcacaactccaggaccactacttggaacgacccaagggtcc
1 occurrences of re1
1 occurrences of re2
the output shows first the sequences that you should order, second the alignment of these
sequences that will produce the gene in question, and third the number of occurrences of
re1 and re2. This output shows that each restriction site occurs once (as specified in
prefix and suffix) and therefore doesn't require further adjustment.
the variables returned are:
oligoout:
a cell array containing sequences of the 5 oligos
seq:
the sequence of the gene that should result from the design (after
PCR)
tm_out:
a list of overlap tms (all should be 54 or 56)
overlapbeg_out: positions in seq where overlaps begin
overlapend_out: positions in seq where overlaps end
Since the design returned 5 oligos, we'll have to design a short anti-sense oligo for the
end.
here's the last two oligos in the design:
gaacgacccaagggtcccaggtgaattcgtgggcg
gatcacaactccaggaccactacttggaacgacccaagggtcc
the residues in blue will give a Tm of 54.
this:
gcccacgaattcacctg
The oligo must be the reverse complement of
note: there is a flaw in my example… the final anti-sense oligo extends beyond the
constant suffix sequence, which means that it will be different for every gene. This is
fine for a single gene, but if you're making a family of genes, they might as well use
the same anti-sense oligo, so just make the suffix longer.
KRANTZ LAB
Version 3
makeoligos2bv3.m
Function:
[oligoout,seq,tm_out,overlapbeg_out,overlapend_out]=makeoligos2bv3(protein,prefix,suffix,
readingframe,org,re1,re2,re3,re4);
Example:
[oligoout,seq,tm_out,overlapbeg_out,overlapend_out]=makeoligos2bv3('PLPPGWEIKIDPQTGWPFFVD
HNSRTTTWNDPRVP','actgacggatcc','ggtgaattcgtgggcg',1,'e','ggatcc','gaattc','agatct','ctcga
g');
Note ‘org’ moved up in the parameter list and now 4 restriction sites may be added to
search for. Requires an additional file: getrestrictionsitename.m
Other restriction sites may be added to the above function.
Bryan
6/16/2008 4:47 PM
U
C
A
G
E. coli prefs
U
Phe
UUU
Phe
UUC
Leu
UUA
Leu
UUG
Leu
CUU
Leu
CUC
Leu
CUA
Leu
CUG
Ile
AUU
Ile
AUC
Ile
AUA
Met
AUG
Val
GUU
Val
GUC
Val
GUA
Val
GUG
G
A
C
U
yeast prefs, from
U
Phe
UUU
Phe
UUC
Leu
UUA
Leu
UUG
Leu
CUU
Leu
CUC
Leu
CUA
Leu
CUG
Ile
AUU
Ile
AUC
Ile
AUA
Met
AUG
Val
GUU
Val
GUC
Val
GUA
Val
GUG
57
43
13
13
10
10
4
50
51
42
7
100
26
22
15
37
C
Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
A
Tyr
Tyr
Stop
Stop
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
ftp://genome-ftp.stanford.edu/pub/codon/ysc.gene.cod
C
A
G
58 UCU Ser 27 UAU Tyr 56 UGU Cys
64
42 UCC Ser 16 UAC Tyr 44 UGC Cys
36
28 UCA Ser 21 UAA Stop 50 UGA Stop 28
30 UCG Ser 9 UAG Stop 22 UGG Trp 100
12 CCU Pro 31 CAU His 64 CGU Arg
15
5
Pro 15 CAC His 36 CGC Arg
5
CCC
14 CCA Pro 42 CAA Gln 70 CGA Arg
6
11 CCG Pro 12 CAG Gln 30 CGG Arg
4
47 ACU Thr 35 AAU Asn 59 AGU Ser
16
27 ACC Thr 22 AAC Asn 41 AGC Ser
11
26 ACA Thr 30 AAA Lys 57 AGA Arg
49
100 ACG Thr 13 AAG Lys 43 AGG Arg
21
40 GCU Ala 38 GAU Asp 65 GGU Gly
49
21 GCC Ala 23 GAC Asp 35 GGC Gly
19
20 GCA Ala 28 GAA Glu 71 GGA Gly
21
19 GCG Ala 11 GAG Glu 29 GGG Gly
11
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
57
43
65
6
57
43
35
65
45
55
77
23
63
37
69
31
G
Cys
Cys
Stop
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
44
56
29
100
38
40
7
10
15
28
4
2
34
40
11
15
UCU
15
15
12
15
16
12
19
53
17
43
13
27
16
27
21
36
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG